Methods for processing biological tissue

ABSTRACT

A method for processing biological tissue used in biological prostheses includes providing a tissue procurement solution formed from a phosphate buffered saline (PBS) solution and a chelating agent. The tissue is transferred from the tissue procurement solution and undergoes chemical fixation. The fixed tissue is then immersed in a series of fresh bioburden reduction process (BRP) solutions to extract phospholipids. The tissue procurement solution reduces the bioburden on the stored tissue and preserves tissue architecture by minimizing tissue swelling. The tissue procurement solution further reduces calcium from the incoming water and/or tissue, and inhibits enzymes that digest the collagen matrix. The serial immersion of the tissue in the fresh bioburden solutions ensures optimal extraction of phospholipids thereby mitigating subsequent calcification of the tissue.

FIELD OF THE INVENTION

The present invention relates generally to biological prostheses, and inparticular, to methods for processing biological tissue that are used inbiological prostheses, such as heart valves.

BACKGROUND OF THE INVENTION

Biological prostheses or “bioprostheses” are devices derived at leastpartially from processed biological tissues to be used for implantationinto humans. Examples of bioprostheses that are currently used or indevelopment include heart valves, vascular grafts, ligament substitutes,pericardial patches, and others. Even though much is now known aboutbiological tissue, bioprostheses and the processing, assembly, andperformance thereof, there are still deficiencies that need to beovercome to provide a bioprosthesis that preserves the native tissueproperties while optimizing tissue biomechanics, minimizingcalcification, and/or rendering the treated tissue hemocompatible.

For example, the biological tissue that is harvested from a donor mustbe stored under proper conditions, and in proper solutions, to preservethe native properties of the tissue prior to and during the tissueprocessing steps that are to be subsequently undertaken. In addition,the harvested biological tissue should be stored in a manner thatmitigates or even reduces the bioburden of the harvested tissue.

Further, the primary component of biological tissues used to fabricatemany bioprostheses is collagen, a term used herein a generic sense torefer to a family of related extracellular proteins. Collagen moleculesassemble to form microfibrils, which in turn assemble into fibrils,resulting in collagen fibers. The amino acids that make up the collagenmolecules contain side groups that represent sites for potentialchemical reaction on these molecules. Because collagenous tissuesdegrade rapidly upon implantation into a host recipient, it is necessaryto stabilize the tissue if it is to be used for long-term clinicalapplications. Chemical stabilization by cross-linking collagen moleculeswithin the tissue (also known as tissue fixation) is well-known, andglutaraldehyde is commonly used to cross-link tissue.

Unfortunately, glutaraldehyde-fixed bioprosthetic tissues tend to becomecalcified over time. The mechanism by which calcification occurs inglutaraldehyde-fixed bioprosthetic tissue has not been fully explained,and many factors have been thought to influence the rate ofcalcification. In general, the calcification phenomenon has beencharacterized as being due to intrinsic causes (i.e., causes inherentlycontained within the tissue itself) and extrinsic causes (i.e., causesfrom outside the tissue itself, such as infection, patient's age,existing metabolic disorders, flow disturbances, etc.). One intrinsiccause of calcification has been shown to be the presence ofphospholipids in the harvested tissues. See e.g., Cunanan et al., Tissuecharacterization and calcification potential of commercial bioprostheticheart valves, Annals Thoracic Surgery, 2001; 71:S417-S421. Therefore, itis desirable to mitigate or inhibit the calcification of the tissue inorder to increase the usable life of any bioprosthesis that is implantedinto a human host.

SUMMARY OF THE INVENTION

The present invention relates to apparatus, solutions, and methods forcollecting, shipping and/or processing fresh tissue, such as from theabattoir. In one embodiment, the solutions and methods may inhibitenzymatic degradation of the tissue matrix during shipment, suppressesor inhibits microbiological activity and growth prior to fixation,and/or removes background levels of calcium in the harvested tissue andwater.

Furthermore, a series of solution buffers may be provided that areosmotically well-balanced and optimized to provide a greater bufferingcapacity and longer product shelf-life. The solutions and methods mayalso provide a tissue that more closely retains the native water contentof pericardial tissue compared to other processes, thereby preservingthe tissue's natural state prior to fixation.

In another embodiment, the tissue may be repeatedly and consecutivelytreated with a calcification mitigant to substantially removecalcification initiators from the tissue, thereby removing an intrinsicmechanism for calcification and reducing the possibility of early valvefailure. This also may further improve the long-term durability of theresulting valve. Moreover, the tissue surface may be renderedhemocompatible during the overall process, increasing the hydrophilicityof the tissue surface compared to glutaraldehyde alone or fresh tissue.

In yet another embodiment, a solution is provided for storing biologicaltissue used in a biological prostheses that includes a mixture ofphosphate buffered saline (PBS) solution having a concentration of atleast 50 mM and a chelating agent. In another embodiment, a solution forstoring biological tissue used in a biological prostheses includes amixture of phosphate buffered saline (PBS) solution having aconcentration of at least 100 mM and a chelating agent.

In still another embodiment, a solution is provided for storingbiological tissue that is used in biological prostheses that includes amixture of a chelating agent and a buffer selected from the groupconsisting of a phosphate-based buffer, a citrate-based buffer, and aborate-based buffer, the buffer having a concentration of at least 100mM.

In yet another embodiment, a method is provided for storing harvestedtissue used in biological prostheses that includes providing a solutioncontaining a mixture of phosphate buffered saline (PBS) solution havinga concentration of at least 100 mM and a chelating agent, and immersingthe harvested tissue in the solution.

In another embodiment, a method is provided for processing fixedbiological tissue that includes: (a) immersing the fixed biologicaltissue in a first fresh bioburden reduction process (BRP) solution; (b)heating the first fresh BRP solution to a temperature of about 37° C.;(c) removing the fixed biological tissue from the fresh BRP solution;(d) immersing the fixed biological tissue in a next fresh BRP solution;and (e) heating the next fresh BRP solution to a temperature of about37° C. Optionally, steps (c), (d), and (e) may be repeated a pluralityof times.

In still another embodiment, a method is provided for processingbiological tissue that includes: immersing tissue in a mixture ofphosphate buffered saline (PBS) solution having a concentration of atleast 50 mM and a chelating agent; transferring the tissue to aphosphate buffered fixation solution; cutting a portion of the tissueinto a desired shape; and transferring the cut tissue portion to aseries of fresh bioburden reduction process (BRP) solutions, whereineach fresh BRP solution is heated for a period of time.

These and other aspects of the invention are described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart of an exemplary tissue processing method.

FIG. 2 is a flowchart of a tissue harvesting procedure that may be usedwith the tissue processing method of FIG. 1.

FIG. 3 is a flowchart of a process that may be used to receive harvestedtissue in accordance with the tissue processing method of FIG. 1.

FIG. 4 is a flowchart of a process that may be used to perform a cutoperation in accordance with the tissue processing method of FIG. 1.

FIG. 5 is a flowchart of a process that may be used to select tissuesections in accordance with the tissue processing method of FIG. 1.

FIG. 6 is a flowchart of a process for forming leaflet-laminatesub-assemblies and reducing the in-process bioburden on them that may beused with the tissue processing method of FIG. 1.

FIG. 7 is a flowchart of a process for assembling a prosthetic heartvalve that may be used with the tissue processing method of FIG. 1.

FIG. 8 is a flowchart of a process for testing a prosthetic heart valvethat may be used with the tissue processing method of FIG. 1.

FIG. 9 illustrates reduction in bioburden levels in tissue that has beenstored in a tissue procurement solution prepared as described herein.

FIG. 10(a) illustrates a radiograph of a sample of fixed tissue that hasundergone processing as described herein.

FIG. 10(b) illustrates a radiograph of a control sample of fixed tissue(no BRP—glutaraldehyde only).

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 illustrates an exemplary method for processing tissue. Eventhough the method is described in connection with harvesting andprocessing bovine pericardium tissue used to construct heart valves,these principles may also be applied to other types of biological tissueand/or for constructing other bioprostheses. Examples of other types ofbiological tissue include porcine, bovine, ovine, or other aortic orpulmonary valves and vascular tissues; human donor allografts; othersources of connective tissue matrices, including porcine, equine, ovineand other xenogeneic or allogeneic pericardial tissues; dura mater;omentum or other tissues of the digestive tract; skin, placenta, uterus,or tissues reconstructed in vitro from cells from such tissues; andocular tissues including cornea and sclera.

Examples of other bioprostheses or devices that may be formed frombiological tissue processed as described herein include heart valves andvalve leaflets; vascular grafts for peripheral, coronary and dialysisassess; patches, strips, or buckles used to reinforce or repair softtissues, hard tissues, cartilage, tendon, cornea, or the like for organrepair and reinforcement for effective reconstruction procedures(including native valve reconstruction, valve annuloplasty and repair).The methods may also be applied to create structures or devices fortissue augmentation procedures (including cardiac wraps, bands, orreinforcements for congestive heart failure, vascular aneurysm repairand reinforcement including cerebral, aortic, and abdominal devices),and as an adjunct or support for other devices fabricated from syntheticmaterials such as DACRON or PTFE; and scaffolds for repairing and/orregenerating tissues, either before or after implantation.

With reference to FIG. 1, in step 10, the tissue is harvested at aslaughterhouse, abattoir, or the like using known techniques, and theharvested tissue is transferred to a container such as, a shippingcontainer that contains a tissue procurement solution, such as thatdescribed further below. The shipping container is then delivered to theassembly location.

FIG. 2 illustrates a method for harvesting biological tissue for use ina biological prosthesis. As seen in FIG. 2, the source of tissue (e.g.,bovine) is received and inspected (step 10 a). Once the source of tissueis deemed suitable for human consumption (step 10 b), the source iskilled and is hung and skinned (step 10 c). Next, the offal is isolatedand inspected (step 10 d). The heart sac or pericardium is removed fromthe heart organ and is stored on ice (step 10 e). Next, in step 10 f,the pericardia is then transferred to a processing area where the fatpad is removed. The processed tissue is then rinsed one or more times,e.g., three times in a rinse saline solution as indicated in step 10 g.The tissue is then transferred to a shipping container or the like,e.g., containing the tissue procurement solution described in detailbelow (step 10 h). The tissue contained in the tissue procurementsolution may be packed on ice for the duration of the transfer. Finally,the shipping container is transferred from the abattoir to a processinglocation, e.g., via overnight courier (step 10 i).

Referring back to FIG. 1, in step 12, the tissue is then received andwashed using standard techniques. FIG. 3 illustrates one method forreceiving and washing tissue. As seen in FIG. 3, in step 12 a, thetissue is first received at the processing location. The shipment may beinspected in step 12 b and transferred to a biohazard hood (step 12 c).The tissue is then washed in PBS one or more times, e.g., three times,and is transferred to a PBS storage solution (step 12 d). Finally, thetissue may be stored in a closed container until the tissue is ready forfurther processing (step 12 e).

As is seen in step 14 of FIG. 1, the washed tissue may then betransferred to a clean room for a cut procedure. FIG. 4 illustrates thesteps involved in an exemplary cut procedure. As seen in step 14 a inFIG. 4, the outside of the container may be wiped down and transferredto a clean room. Next, in step 14 b, the container may be placed in afume hood and the work area may be prepped. In step 14 c, the pericardiais removed from the container (one at a time) and a cut is made of thetissue. Next, the cut tissue is transferred for tissue fixation (step 14d).

Referring back to FIG. 1, in step 16, the cut tissue may then be subjectto tissue fixation and quarantine. First, the tissue may be hung inglutaraldehyde solution, e.g., as is described further below. Afterbetween about thirty minutes to twenty four hours exposure to theglutaraldehyde solution, the tissue may be removed from the fixationsolution and placed into a storage/quarantine container, e.g., withfresh glutaraldehyde solution. The glutaraldehyde solution may bereplenished periodically, for example, once every three to four daysduring storage/quarantine.

If desired, the tissue may be stored in a tissue bank until needed. Thisoptional step is shown, for example, in FIG. 1 as step 17.

Next, in step 18 shown in FIG. 1, the good tissue sections (i.e., thosetissue sections having desirable properties for a prosthetic device) maybe selected, and then leaflets cut in step 20. FIG. 5 illustrates anexemplary process for selecting tissue sections. A piece of tissue mayfirst be removed from the glutaraldehyde storage/quarantine solution andplaced on a light box, inspection table, or tray (step 18 a). Next, asseen in step 18 b, the tissue may be examined for defects such as, largeblood vessels, tears, fatty deposits, excessively thin or thick spots,and the like. Fatty deposits and large strands may then be removed usingtools such as toothless forceps or curved scissors and a scalpel (step18 c). The tissue is now ready for cutting, e.g., as described withrespect to step 20. Optionally, the selection step 18 may also includethe additional steps of using a sterile scalpel to cut out one or moredesirable areas that are large enough for a leaflet (step 18 d). Thecut-out areas may then be stored in a glutaraldehyde solution until theleaflets are ready to be cut (step 18 e).

Referring back to FIG. 1, in step 22, sub-components of the final valvemay be assembled. For example, for a bovine pericardial tri-leafletvalve, each leaflet may be stitched or otherwise attached to a laminateor frame to form one of three sub-components or leaflet sub-assembliesin step 22. The leaflet sub-assemblies may also include othercomponents, for example, a fabric covering at least a portion of thelaminate or frame, one or more connectors, and the like. As used herein,the words “sub-component” and “sub-assembly” may be usedinterchangeably, and have the same meaning. Additional information onleaflet sub-assemblies, and methods for making them may be found in U.S.Pat. No. 6,371,983, the entire disclosure of which is expresslyincorporated by reference herein.

Turning to FIG. 6, an exemplary method is shown for making one or moreleaflet sub-assemblies for a valve assembly of a one-piece or multiplecomponent heart valve prosthesis. As seen in step 22 a, the non-tissuecomponents may first be cleaned, e.g., in isopropyl alcohol (IPA). Forthe tissue components, the glutaraldehyde solution may be drained fromthe leaflets (step 22 b). The leaflet sub-assembly may then be formed.As seen in FIG. 6 (step 22 c), after the proper size and quantity oflaminates/leaflets is confirmed, the individual leaflets are sewn torespective laminates.

With reference now to FIGS. 1 and 6, the resulting leaflet sub-assemblymay then be immersed in a bioburden reduction process (BRP) solution(step 24 in FIG. 1; step 24 a in FIG. 6), e.g., as described in moredetail below. Generally, this involves heating the sub-assembly in theBRP solution for a period of time between about four and twenty-four(4-24) hours at a temperature of around thirty-seven degrees Celsius(37° C.), e.g., at a ratio of about one hundred milliliters per valve(100 mL/valve) equivalent. In one embodiment, the BRP solution may bedrained off the leaflet sub-assembly one or more times, and the leafletsub-assembly may be immersed in fresh BRP solution (step 24 b in FIG.6). Incubation is continued at a temperature of around 37° C. for atotal wash time of about twenty-four (24) hours.

Next, the leaflet sub-assembly may be subjected to a series of rinses ina glutaraldehyde/PBS solution (step 24 c in FIG. 6). After the rinseprocess, the leaflet sub-assembly may be transferred to a storagecontainer, e.g., having a fresh glutaraldehyde/PBS solution therein. Theleaflet sub-assembly may be stored until future use (step 24 d in FIG.6).

Referring to FIG. 1, in step 26 the leaflet sub-assemblies may beassembled into a valve assembly (i.e., prosthetic heart valve). As seenin FIG. 7, in one embodiment, this process may include assembling theleaflet sub-assemblies to a valve frame(step 26 b) and subsequentlyinspecting the valve assembly (step 26 c).

After assembly, the valve may be tested in step 28 using any desiredtesting methods. Typically, as seen in the process steps shown in FIG.8, these tests may include coaptation tests and forward and/or backwardflow tests. Tests may be performed to ensure that the assembled valvesopen with minimal effort, close with minimal leakage, and/or providesuitable hydrodynamic performance, e.g., at a wide range of operatingflow conditions. The assembled heart valves may also be visuallyinspected in step 30.

Finally, in step 32, the assembled heart valves that pass the tests maybe transferred to a final container where they are subjected to aterminal liquid sterilization (TLS) process before shipment to hospitalsfor implantation by surgeons. A technician or other qualified person mayverify that the necessary documentation is complete and acceptable. Thevalve may then be transferred to a jar or other final container, whichmay be filled with a terminal sterilant. Next, a seal may be placed overthe jar and sealed in place. The valve may be sterilized in the jar,e.g., by heating the jar to a desired temperature for an extended periodof time. A final jar inspection may be performed to ensure the integrityof the package and any labels thereon. Finally, the valve may then beplaced in storage until use.

During the various steps described herein, solutions may be used toensure long shelf-life and/or maximal buffering capacity. Thesesolutions may include optimal formulations that include high phosphates,including PBS-based solutions, which provide greater bufferingcapability, e.g., to protect the tissue against the inherent property ofglutaraldehyde solutions to become more acidic with time.

For example, a tissue procurement solution may be used to store tissueafter harvesting and/or before tissue fixation (i.e., after step 10 inFIG. 1). The tissue procurement solution may use an osmotically-balancedbuffered salt solution, which may better preserve tissue structureand/or minimize tissue swelling. The tissue procurement solution mayalso include a chelating agent to chelate calcium and divalent cations.Chelating calcium may aid in removing adherent cells without damagingthe tissue, may reduce calcium from the incoming water/endogenous levelsin tissue, and/or may inhibit enzymes that may be released during tissueshipment or storage, which tend to digest and degrade the collagenmatrix. The chelating agent may also interfere with microbial activity,thereby reducing the bioburden on the stored tissue.

In one embodiment, the tissue procurement solution may be composed of abuffering agent with a concentration of at least about 25 mM. In anotherembodiment, the buffering agent may have a concentration of at leastabout 50 mM. In yet another embodiment, the buffering agent may have aconcentration of at least about 100 mM. The buffering agent may providesuitable buffering capacity around pH 6-8, or around pH 7, or within therange of pH 7.3-7.5. The buffering agent may be chemically inert withrespect to glutaraldehyde reactivity, and so buffers, such as HEPES andTRIS, may be unsuitable in this application. Exemplary buffers includephosphate-based buffers, citrate-based buffers, and borate-basedbuffers. Phosphate buffers may be particular useful due to their readysupply, strong buffering capacity, and compatibility with downstreamprocessing chemicals.

The solution may also contain a chelating agent, generically describedas a chemical agent to complex or to bind divalent cations. It is highlydesirable in the procurement of fresh tissues to inhibit the action ofproteolytic enzymes before preserving the tissue by fixation techniques,and the binding of divalent cations “stops” or otherwise inhibits theaction of many enzymes that require divalent cations in the active sitefor proteolysis. Divalent cations are also essential for basic cellularfunctions, such as adhesion and cell division, so chelation of divalentcations may also be an effective way to remove adherent cell layers withminimal handling. Bacterial cells may also be inhibited by the chelationof divalent cations, so that the use of chelating agents in theprocurement solution of fresh tissues may aid in bacteriostasis.

It has been found that the tissue procurement solution has the abilityto maintain or even reduce the bioburden on harvested tissue. FIG. 9,for example, illustrates the reduction in bioburden levels in tissuethat has been stored in the above-described tissue procurement solution.Finally, with respect to the chelating aspect of the tissue procurementsolution, by chelating divalent cations, including divalent cations suchas calcium ions, the overall calcium load in tissue may be reduced, bothfrom the tissue itself and from processing chemicals and water.

Suitable chelation agents that may be used in the tissue procurementsolution include well-known aminopolycarboxylic acids, such as EDTA(ethylenediaminetetraacetic acid) and EGTA(bisaminoethyl-glycolethertetraacetic acid), as well as polymeric etherchelation agents such as the polyoxyethylenes, polyoxyglycols, andpoly-glymes; other structural components which form similar shapes suchas cyclic antibiotics, amino acid peptides, and wholly synthetic orbiological compounds, such as modified fullerenes, dendrimers,polysaccharides, polynucleic acids, or other compounds capable ofcomplexing divalent cations due to their three dimensional shape andionic character. The principal action of the agents described iscomplexation of metal compounds, such as calcium and magnesium, throughone or more electron-donating groups. Metal cations have severalavailable orbitals for bond formation with complexing agents; therefore,the chelating agent can be monodentate (from the Latin word dentatus,meaning “toothed.”), such as the chlorides, cyanides, hydroxides, orammonia complexes, and mixed complexes may be formed from these. Inaddition, the ligand may be multidentate, or containing multiple teeth,which can contribute two or more electron pairs to a complex.Ethylenediamine, NH₂CH₂CH₂NH₂, is an exemplary bidentate ligand. Otheruseful members of the aminopolycarboxylic acid family include DCTA(trans-diaminocyclohexanetetraacetic acid), NTA (nitrilotriacetic acid),and DTPA (diethylenetriameinepentaacetic acid).

In one embodiment, the tissue procurement solution may be delivered orotherwise formed on-site in concentrate form that may be subsequentlydiluted, e.g., before tissue submersion. Table 1 below illustrates anexample of a solution of a PBS-EDTA tissue procurement solution. Afterdilution to one liter (1.0 L), the tissue procurement solution has a pHwithin the range of about 7.3 to 7.5. The final pH of the solution maybe adjusted with either 1N hydrochloric acid (HCl) or 1N sodiumhydroxide (NaOH) on an as-needed basis. In addition, post-dilution, thetissue procurement solution may have an osmolarity level within therange of about 290-310 mOsm or around 300 mOsm. TABLE 1 ChemicalSpecification Sodium Chloride (NaCl) 20.4 ± 0.1 g Potassium PhosphateMonobasic 20.0 ± 0.1 g (KH₂PO₄) Sodium Phosphate Dibasic  230 ± 0.1 gheptahydrate (Na₂HPO₄.7H₂O) EDTA 25.0 ± 0.1 g Hydrochloric Acid (HCl) Asneeded Sodium Hydroxide (NaOH) As needed Purified Water Dilute to 1.0 L

Tissue stored in the tissue procurement solution described above may beremoved and subjected to a fixation process (i.e., step 16 in FIG. 1).In one embodiment, the fixation method involves the application of adirectional force on a piece of tissue during the fixation process. Themethods and apparatus disclosed in Lee et al., The Bovine PericardialXenograft: III. Effect of Uniaxial and Sequential Biaxial Stress DuringFixation on the Tensile Viscoelastic Properties of Bovine Pericardium,Journal of Biomedical Materials Research, Vol. 23, 491,-506 (1989) maybe used to apply the directional force. The above-identified Lee et al.publication is expressly incorporated by reference as if set forthherein.

In an alternative embodiment, the tissue strips may be pre-conditionedin a phosphate-buffered saline (PBS) solution for about thirty minutesat room temperature. Pre-conditioning may be desired in processes thatseek minimal tissue fixation durations and/or utilize tissues that arenot easily aligned with a directional force. In such cases,pre-conditioning enables a relaxation of the tissue componentarchitecture under the directional force before application of fixationchemicals, and may be enhanced with suitable time, temperature, and/orvariable weights.

The fixation solution may be a glutaraldehyde solution or any otherknown fixation solution. In one embodiment, the fixation solution mayinclude between about 0.50% and 0.65% glutaraldehyde (on a volume basis)in PBS. In another embodiment, the fixation solution may include about0.57% glutaraldehyde (on a volume basis) in PBS buffer. Table 2 belowillustrates an example of a 0.57% glutaraldehyde solution. Afterdilution to one liter (1.0 L), the solution has a pH within the range ofabout 7.3 to 7.5. The final pH of the solution may be adjusted witheither 1N hydrochloric acid (HCl) or 1N sodium hydroxide (NaOH) on an asneeded basis. In addition, post-dilution, the tissue procurementsolution has an osmolarity level within the range of about 290-310 mOsmor around 300 mOsm. TABLE 2 Chemical Specification Sodium Chloride(NaCl) 0.96 (as needed) Potassium Phosphate Monobasic  2.00 ± 0.02 g(KH₂PO₄) Sodium Phosphate Dibasic 20.57 ± 0.02 g heptahydrate(Na₂HPO₄.7H₂O) 24% Glutaraldehyde 23.75 ± 0.1 ml Hydrochloric Acid,1N(HCl) As needed Sodium Hydroxide, 1N(NaOH) As needed Purified WaterDilute to 1.0 L

After fixation, the tissue is then placed into a glutaraldehyde solutionfor quarantine/storage. For example, the tissue strip may be laid flat,e.g., with the smooth side facing upwards, in a covered sterile holdingtray that is filled with sterile filtered 0.57% glutaraldehyde and PBS.This glutaraldehyde solution may be replenished periodically (e.g.,every three to four (3-4) days) during quarantine. Quarantine is theperiod of time that allows the fixation reaction to go substantially tocompletion. After the fixation quarantine time is substantiallycomplete, tissues may be stored for long periods of time, e.g., untilneeded, particularly if the tissues are refrigerated, for example,stored in a liquid media at a temperature within the range of about twoto twelve degrees Celsius (2-12° C.) or similar chilled conditions.

Optionally, as an alternative to the application of a uniaxial(one-directional) force, it is also possible to apply a biaxial(two-directions) or an isometric (all directions) force by positioningweight(s) at the desired portions of the tissue strip.

During the process of fixation, application of a force in a longitudinaldirection along the length of the tissue is believed to align thecollagen molecules during fixation with crosslinking agents, such asglutaraldehyde. The tissue is suspended in crosslinking solution, withone end fixed to a holder, and the other end left free with a hangingweight. Applying a biasing force to the tissue during fixation may alsoproduce more uniform tissue by stretching out some of the intrinsicvariability from piece to piece, which may facilitate manufacturing bycreating substantially uniform tissues with predictable properties.Thus, this fixation method may enable the engineering of tissue withspecific biomechanical properties, while substantially reducingtissue-to-tissue variability.

After the fixation process, tissue sections may be selected and cut intoone or more desired shapes or geometries, e.g., by die-cutting or lasercutting. In the case of prosthetic heart valves, the tissue may be cutinto leaflets.

The solutions and procedures described herein may provide apost-fixation method that mitigates or inhibits calcification of tissue.An exemplary post-fixation treatment method involves repeatedlyimmersing the fixed tissue in a series of fresh bioburden reductionprocess (BRP) solutions to effect more complete extraction ofphospholipids. This method may be applied to tissue alone, or, as shownin step 24 of FIG. 1, to the tissue and its non biological components,e.g., leaflet sub-assemblies.

In one embodiment, the BRP solution may be a glutaraldehyde andpolysorbate-80 solution (i.e., TWEEN-80), such as that described in U.S.Pat. No. 4,885,005 (Nashef et al.), the entire disclosure of which isexpressly incorporated by this reference herein. For example, the BRPsolution may be an aldehyde-polysorbate-80 solution solution, e.g., asdescribed in Example V of U.S. Pat. No. 4,885,005 (Nashef et al.).

Table 3 below illustrates an exemplary BRP solution. After dilution toone liter (1.0 L), the BRP solution has a pH within the range of about7.3 to 7.5. The final pH of the solution may be adjusted with either 1Nhydrochloric acid (HCl) or 1N sodium hydroxide (NaOH) on an as neededbasis. TABLE 3 Chemical Specification Sodium Chloride (NaCl) 2.00 gPotassium Phosphate Monobasic 2.24 g (KH₂PO₄) Sodium Phosphate Dibasic23.54 g heptahydrate (Na₂HPO₄.7H₂O) 24% Glutaraldehyde 41.67 mlPolysorbate 80 (Polyoxyethylene 20.0 ml Sorbitan Monoleate;polysorbate-80) Hydrochloric Acid, 1N(HCl) As needed Sodium Hydroxide,1N(NaOH) As needed Purified Water Dilute to 1.0 L

According to one post-fixation method (see FIG. 6), the tissue may beplaced in a first container that contains the BRP solution. Thecontainer may be a jar or other vessel, and the tissue may be placedinto the BRP solution retained therein. If the tissue has been assembledinto a sub-component, e.g., as in step 22 of FIG. 1, then the entiresub-component (e.g., tissue, laminate, cloth, etc.) may be placed in theBRP solution inside the container.

Next, in step 24 a, the container (with the tissue or sub-componentimmersed within the BRP solution therein) may be heated for a period oftime at a desired temperature, e.g., of about thirty-seven degreesCelsius (37° C.±2° C.). The container, and consequently, the BRPsolution, may be heated for a period of time between about four andsixteen (4-16) hours. Next, as seen in step 24 b, the tissue orsub-component is then removed from the container, and immediately placedinto a second container that contains a fresh BRP solution. This secondcontainer may then be heated for a period of time at a temperature,e.g., also of about thirty-seven degrees Celsius (37° C.±2° C.). Again,the BRP solution may be heated for a period of time, e.g. between aboutfour and sixteen (4-16) hours. Optionally, the first container may bereused as the second container (as long as the first container isrefilled with a fresh BRP solution).

The above described process may be repeated one or more times in aplurality of fresh BRP solutions. Generally, the process may be repeatedwith any number of fresh BRP solution changes, as long as the total timethe tissue is heated at the desired temperature is at least about one(1) hour. While there is no apparent upper limit to this exposureprocess, from the practical aspects of the process, it may be desirableto limit the overall BRP exposure to about twenty four (24) hours. As anexample, the tissue or sub-component may be heated at the desiredtemperature, e.g., about 37° C. (±2° C.), in a first fresh BRP solutionfor about four (4) hours, in a second fresh BRP solution for about six(6) hours, in a third fresh BRP solution for about four (4) hours, andin a fourth fresh BRP solution for about six (6) hours (total of abouttwenty hours (20) exposure). The effective driving force for theextraction process is the physicochemical partitioning of thephospholipids between the tissue phase and the soluble phase (i.e., BRPsolution). Thus, performing the extraction process at least twice,immediately after the other, is the key to efficiently removing thephospholipids. The process and rate of removal of phospholipids isaffected by several variables including time, temperature, pressure,and/or concentration, and one skilled in the art will know how tooptimize these variables in order to effect optimal extraction.

In an alternative embodiment, the phospholipids may be extracted using aco-current, cross-current, or counter-current separation system whereinthe phospholipids may be substantially continuously extracted with freshBRP solution for maximum extraction efficiency. Such a process may useany combination of time, temperature, concentration, and/or pressure,provided those conditions do not adversely affect the tissue properties.In such an instance, automation and solvent exchange/delivery systemsmay provide manufacturing advantages to help realize the effectivenessof the concept of repeated extractions in removing phospholipids fromtissue to mitigate or reduce calcification.

One skilled in the art will also realize that the composition of the BRPsolution may be varied to include a variety of compounds, as long as thephospholipids are soluble in it. Therefore, while detergents such aspolysorbate-80 are useful, other phospholipid-solvating compounds may beeffective, provided they contain both a polar component and a non-polarcomponent. It is desirable that the polar component be neutral, ratherthan an ionic component. While compounds such as polysorbate-80 containthese functions within the same molecule (known as amphiphilic),mixtures of simple chemicals may achieve the same purpose. For example,mixtures of alcohols with nonpolar compounds (such as ethers,chloroform, or other nonpolar solvents) may be effective; in the case ofwater as the polar compound, alcohols may provide the nonpolarfunctionality of the solution, provided the molecular weight andconcentration of the alcohol in the water are sufficiently high. Forexample, methanol-water mixtures may not be effective solvents forremoving phospholipids in tissues, but ethanol-water mixtures may beused. Generally, the methods described herein contemplate using adetergent or phospholipid-solvating solution comprising multiplesolvents having a wide range of polarities. Temperature, time, pressure,and/or other conditions may again be altered to optimize the removal ofphospholipids. In this particular case, a buffered glutaraldehyde basedsolution (PBS buffer system) may be particularly useful in order toprovide microbiocidal activity while preserving tissue structure(physiologic osmolality, pH, etc.). If, however, it is not desirable ornecessary to retain microbiocidal activity and/or preserve tissuestructure, significantly more leeway may be applied in the choice of thepolar/non-polar system used in the extraction and in the preciseextraction conditions. In particular, processes used to make tissues notperforming significant mechanical functions (soft tissue augmentationand repair, ocular tissues, etc.) may employ a wide range of conditionsin order to accomplish the goals described herein.

After the repeated changes in step 24 b, the tissue or sub-component maybe rinsed in step 24 c. First, the final BRP solution may be drainedfrom the container, and the container filled with a solution, e.g.,containing 0.57% glutaraldehyde and PBS (see above). The tissue orsub-component may be placed into the glutaraldehyde and PBS solution andallowed to sit at a desired temperature, e.g., room temperature, for adesired period of time, e.g., approximately five to ten (5-10) minutes.The container may then be inverted to rinse the tissue or sub-component.This rinsing may be repeated two or more times with two freshglutaraldehyde and PBS solutions, e.g., for a total of three rinses.

After post-fixation treatment, the tissue or sub-component may be storedin a terminal sterilant solution. In one embodiment, the terminalsterilant solution may have anti-microbial properties as well as astrong buffering capacity to ensure longer shelf life. For example, aterminal sterilant solution compliant with International Standard ISO14160 may be used, the entire disclosure of which is expresslyincorporated by reference herein.

EXAMPLE 1

Bovine pericardium tissue samples were subject to the processing stepsdescribed in detail herein. In addition, control samples (“glut only”)were subject to the same processing steps with the exception of thebioburden reduction process and terminal sterilization process. For thecontrol samples, the tissue samples were incubated at 32° C. with a0.57% glutaraldehyde solution.

Samples of bovine pericardium were obtained and stored in a tissueprocurement solution (PBS/EDTA solution) prior to arrival. Initialtissue samples were first rinsed in PBS and fat deposits were removed.The tissue samples were then subject to either uniaxial fixation orisometric fixation. For uniaxial fixation, the tissue was cut into60×160 cm rectangular strips. One end of the tissue (upper end having awidth of 60 cm) was secured to a dialysis clip. A stainless steel rod(weighing about 35-40 g) was placed on the opposing lower end of thetissue strip. The tissue was folded over the rod and secured in placewith a dialysis clip.

A 1000 ml beaker was filled almost to the brim with a 0.57%glutaraldehyde solution. Two stainless steel spatulas were placed inparallel over the top of the beaker. The weight and dialysis clip werethen lowered into the beaker, using the spatulas as a support for thedialysis clip located on the upper end of the tissue. The tissue samplewas allowed to hang immersed in the fixation solution for a minimum ofthirty (30) minutes. After fixation, the tissue clips were removed fromthe sample and the tissue was placed in a storage container filled with0.57% glutaraldehyde solution.

For isometric fixation, bovine pericardium tissue was cut into a 11 cmsquare and placed over the edge of an isometric pressure fixture. Thetop of the pressure fixture is circular in shape and has a diameter ofabout 10 cm. The tissue was fixed to the pressure fixture using a ziptie. A constant flow rate of 0.57% glutaraldehyde solution was thenpumped on the top of the tissue. A hole located about one-quarter inchabove the upper surface of the tissue kept the glutaraldehyde levelconstant during the immersion step.

After fixation, the tissue was cut into either 1×4 cm strips or 10 mmdisks. The rectangular strips were cut using a scalpel while thedisk-shaped samples were formed using either a laser or a conventionaldie cutting process. The cut samples where then subject to a bioburdenreduction process. Control samples were not subject to the bioburdenreduction process. For non-control tissue samples, the tissue was placedin 100 ml jars filled with a bioburden reduction process (BRP) solutionof the type described herein and incubated at 37° C. for sixteen (16)hours. Samples were then removed from the incubator, and the BRPsolution was decanted and replaced with fresh BRP solution. The samplescontinued to incubate at 37° C. for a total of twenty-four (24) hours.

After the incubation period was complete, the jars containing the tissuesamples were rinsed three times using a 0.57% glutaraldehyde solutionfor a period of ten minutes each. A three hour soak of the tissuesamples in 0.57% glutaraldehyde solution followed the rinse steps. Afterthe soak operation, the glutaraldehyde solution was decanted andreplaced with fresh 0.57% glutaraldehyde solution. The samples werestored in this solution at room temperature until ready for the finalsterilization step.

For final sterilization, the tissue samples were removed from thestorage jar and placed into a clean, autoclaved jar. The jar was thenfilled with a terminal sterilant and the jar was closed using a newsterile lid. The jar was then vacuum tested for any leaks. Once the jarwas sealed and no leaks were identified, the jar was placed upright intoa 32° C. incubator for forty-eight hours.

The tissue samples (including controls) were then implantedsubcutaneously in rats. After twenty-one days, the rats were killed andthe tissue was excised and placed in 10% formalin solution. The hosttissue was then removed and the tissue samples were rinsed and dried.The tissue samples were examined for moisture content as well as forcalcium and phosphorous content. Radiograph images were taken of thesamples for analysis of calcium levels. Radiograph photographs indicatedthat the 37 glut only” control samples had higher calcium levels thanboth the strip and disk tissue samples (based on denser appearance ofcontrol radiographs). FIG. 10(a) illustrates a radiograph of a sample ofisometrically fixed tissue that underwent processing according to thepresent invention. FIG. 10(b) illustrates a radiograph of a controlsample of isometrically fixed tissue (no BRP—glutaraldehyde only).

Table 4 below illustrates the measured calcium and phosphorous levelsfor tissue samples subject to isometric fixation. Calcification andphosphorous analysis was performed using Inductively CoupledPlasma—Optical Emission Spectroscopy (ICP-OES). As seen in the Table 4,the control samples showed an average calcium level of 117 μg/mg whilethe tissue samples subject to the bioreduction process showed an averagecalcium level of 0.41 μg/mg. TABLE 4 Arbor Sample Sample Phos- FixationCutting Number Shape Calcium phorus Process Method Method 0304b-1 Disk100 42 Control Uniaxial Scalpel 0304b-1 Disk 116 46 Control UniaxialScalpel 0304b-3 Disk 0.38 2.8 BRP Uniaxial Scalpel 0304b-3 Disk 0.18 2.5BRP Uniaxial Scalpel 0304b-3 Strip 0.74 2.9 BRP Uniaxial Scalpel 0304b-2Disk 113 53 Control Uniaxial Scalpel 0304b-2 Disk 113 58 ControlUniaxial Scalpel 0304b-2 Strip 58 32 Control Uniaxial Scalpel 0304b-4Disk 0.12 2.3 BRP Uniaxial Scalpel 0304b-4 Disk 0.16 2.2 BRP UniaxialScalpel 0304b-4 Strip 0.31 2.4 BRP Uniaxial Scalpel 0304b-5 Disk 122 68Control Isometric Scalpel 0304b-5 Disk 143 82 Control Isometric Scalpel0304b-5 Strip 123 76 Control Isometric Scalpel 0304b-7 Disk 0.18 1.9 BRPIsometric Scalpel 0304b-7 Disk 0.15 2.1 BRP Isometric Scalpel 0304b-7Strip 1.3 8.2 BRP Isometric Scalpel 0304b-6 Disk 115 76 ControlIsometric Scalpel 0304b-6 Strip 108 71 Control Isometric Scalpel 0304b-8Disk 0.79 1.7 BRP Isometric Scalpel 0304b-8 Strip 0.19 2.9 BRP IsometricScalpel Phos- Calcium phorus Glut Only Average - Control (μg/mg) 117.0063.56 Glut Only StDev 21.80 16.79 BRP Process (Uniaxial & Isometric)Average 0.41 2.90 (μg/mg) BRP Process (Uniaxial & Isometric) StDev 0.381.80

While embodiments of the present invention have been shown anddescribed, various modifications may be made without departing from thescope of the present invention. The invention, therefore, should not belimited, except to the following claims, and their equivalents.

1. A solution for storing biological tissue used in a biologicalprostheses comprising a mixture of phosphate buffered saline (PBS)solution having a concentration of at least 50 mM and a chelating agent.2. The solution of claim 1, wherein the PBS solution has a concentrationof at least 100 mM.
 3. The solution of claim 1, wherein the solution hasa pH within the range of 6 to
 8. 4. The solution of claim 3, wherein thesolution has a pH within the range of 7.3 to 7.5.
 5. The solution ofclaim 1, wherein the chelating agent is selected from the groupconsisting of EDTA, EGTA, polyoxyethylene, polyoxyglycol, poly-glyme,cyclic antibiotic, amino acid peptide, fullerene, dendrimer,polysaccharide, and polynucleic acid.
 6. The solution of claim 1,wherein the mixture has an osmolarity value between the range of 290-310mOsm.
 7. The solution of claim 1, wherein the solution reduces thetissue bioburden level upon exposure to the solution.
 8. The solution ofclaim 1, wherein the solution comprises an aqueous mixture of sodiumchloride, potassium phosphate monobasic, sodium phosphate dibasicheptahydrate, and BDTA.
 9. A solution for storing biological tissue thatis used in biological prostheses, comprising a mixture of a chelatingagent and a buffer selected from the group consisting of aphosphate-based buffer, a citrate-based buffer, and a borate-basedbuffer, the buffer having a concentration of at least 100 mM.
 10. Thesolution of claim 9, wherein the selected buffer is chemically inertwith respect to glutaraldehyde. 11-20. (canceled)
 21. The solution ofclaim 1, wherein the chelating agent comprises EDTA.
 22. The solution ofclaim 9, wherein the chelating agent comprises EDTA.